HIV and AIDS remains a persistent problem in the US and around the world. One reason for the lack of better drugs to prevent HIV infection is insufficient understanding of how the virus is released from infected host cells. HIV and other retroviruses depend on host cell factors known as endosomal sorting complex required for trafficking (ESCRT) machinery for the critical step of membrane scission necessary for budding and release. The virally encoded Gag protein assembles on plasma membranes and produces the necessary curvature to package the viral genome. Gag also has specific motifs to bind to ESCRT-I and/or Alix, which can recruit ESCRT-III proteins. ESCRT-III proteins assemble into filaments on membranes to facilitate scission and release virus. However, the molecular mechanisms responsible for ESCRT-III recruitment and activation to release assembled virions remain unclear. In this proposal, we will use deep-etch electron microscopy (EM) in combination with correlative light microscopy to study the relationship between HIV Gag and the cellular ESCRT machinery. Deep-etch EM is a powerful technique for visualizing cellular membranes and membrane surface proteins and can provide nm resolution views of the protein machinery involved in viral particle assembly. We will apply this to (i) define the molecular architecture of connections between HIV Gag and ESCRTs stabilized by the absence of Vps4 and (ii) together with correlative light microscopy extend these studies to examine transient ESCRT structures present during normal viral particle assembly. Results from these experiments will move understanding of HIV and other retrovirus budding forward, paving the way for developing new strategies to intervene in HIV infection.